Graves’ disease is an autoimmune disorder caused by auto-antibodies against the thyroid stimulating hormone receptor (TSHR). Overstimulation of the TSHR induces hyperthyroidism and thyroid eye disease (TED) as the most common extra thyroidal manifestation of Graves’ disease. In TED, the TSHR cross talks with the insulin-like growth factor 1 receptor (IGF-1R) in orbital fibroblasts leading to inflammation, deposition of hyaluronan and adipogenesis. The bone marrow may play an important role in autoimmune diseases, but its role in Graves’ disease and TED is unknown. Here, we investigated whether induction of experimental Graves’ disease and accompanying TED involves bone marrow activation and whether interference with IGF-1R signaling prevents this activation.in vitro prevented immune-inhibition by linsitinib.Immunization of mice with TSHR resulted in an increase the numbers of CD4-positive T-lymphocytes (p ≤0.0001), which was normalized by linsitinib (p = 0.0029), an increase of CD19-positive B-lymphocytes (p= 0.0018), which was unaffected by linsitinib and a decrease of GR1-positive cells (p= 0.0038), which was prevented by linsitinib (p= 0.0027). In addition, we observed an increase of Sca-1 positive hematopietic stem cells (p= 0.0007) and of stromal cell-derived factor 1 (SDF-1) (p ≤0.0001) after immunization with TSHR which was prevented by linsitinib . TSHR-immunization also resulted in upregulation of CCL-5, IL-6 and osteopontin  and a concomitant decrease of the immune-inhibitory cytokines IL-10 (p= 0.0064) and PGE2 (p ≤0.0001) in the bone marrow . Treatment with the IGF-1R antagonist linsitinib blocked these events . We further demonstrate a down-regulation of arginase-1 expression (p= 0.0005) in the bone marrow in TSHR immunized mice, with a concomitant increase of local arginine (p ≤0.0001). Linsitinib induces an upregulation of arginase-1 resulting in low arginase levels in the bone marrow. Reconstitution of arginine in bone marrow cells Collectively, these data indicate that the bone marrow is activated in experimental Graves’ disease and TED, which is prevented by linsitinib. Linsitinib-mediated immune-inhibition is mediated, at least in part, by arginase-1 up-regulation, consumption of arginine and thereby immune inhibition. FemaGD is an autoimmune disease of the thyroid which is caused by autoantibodies against the thyroid stimulating hormone receptor (TSHR) (TSHR Ab = TRAb), leading to an overproduction of thyroid hormones, overstimulation of the thyroid gland and consecutive hyperthyroidism , 5. ThyrAnother important receptor in the pathogenesis of TED is the insulin-like growth factor 1 receptor (IGF-1R) which has come more into focus in mediating the disease progression and is known to be overexpressed by thyroid follicular cells and OF’s as well as immune cells such in patients suffering from Graves’ disease and TED –11. In rLinsitinib inhibits the autophosphorylation of the IGF-1R and therefore blocks important downstream signaling pathways, such as AKT and ERK signaling .early and late phase of the autoimmune disorder in target tissues and also downregulated the inflammation in the orbital tissue, indicating the clinical significance of our findings.We have recently demonstrated that linsitinib, also known as OSI-906, a selective small-molecule dual inhibitor of the insulin like growth factor 1 receptor (IGF-1R) and insulin receptor (IR) , has a mRecently Shi et. al., documented in an experimental autoimmune encephalomyelitis (EAE), an established mouse model for multiple sclerosis, that autoreactive T-cells migrate into the bone marrow and stimulate hematopoietic stem and progenitor cells to differentiate towards myeloid lineages that mediate inflammatory brain injury in the model . The autSimilar to multiple sclerosis, TED is mainly a T-cell-, especially a Th1 and TH17 mediated autoimmune disease , 21 and Here, we investigated in an experimental murine model of Graves’ disease, whether autoimmune GD and TED associates with bone marrow activation and whether linsitinib, an oral small molecule inhibitor of the IGF-1R and insulin receptor (IR), interferes with this activation process. In particular, we investigated whether immunization with TSHR results in (i) a change of the cellular components of the bone marrow, (ii) a potential recruitment or efflux of immune cells and (iii) a proliferation of stem cells, (iv) a release of pro-inflammatory cytokines driving an immune response and (v) a reduction of anti-inflammatory cytokines and the expression of arginase, which would also drive an immune response. We tested whether linsitinib has an effect on these changes of the bone marrow upon immunization with TSHR.22.1Female BALB/c inbred mice were purchased from Envigo Netherlands GmbH and housed under specifically pathogen-free conditions as described before . Mice wend group, we treated eight weeks old male C57BL/6 mice with linsitinib and also isolated bone marrow cells from these mice. The data obtained with these mice are displayed in panel 7. We used two different mouse strains to confirm the principal findings in an independent mouse strain.In a 2All animal procedures were approved by the North Rhine Westphalian State Agency for Nature, Environment and Consumer Protection (LANUV), Germany, or by the local IACUC, University of Cincinnati, Cincinnati, USA. All experiments were performed according to the FELASA regulations and ARRIVE guidelines.2.2We employed 10 animals/group. Specific immunization was obtained by immunizing mice with the eukaryotic expression plasmid pTriEx1.1Neo-human driving expression of the (h)TSHR A-subunit . Control mice were immunized with the pTri1Ex1.1Neo-ß-Gal plasmid, encoding non-immunogenic ß-Gal  as previously described . We immuNone of the mice became thyrotoxic as determined by the already reported T4 levels in, the histology of the thyroid and the daily checked health status of these mice .Linsitinib in the BALB/c mice was applied by daily gavage as previously described . The conThe exact timing of the experiment was chosen due to several other experiments our group performed with the BALB/C mice strain in order to induce Graves’ Disease and TED –27. In t2.32 and arginase-1 concentrations. ELISA assays were performed exactly following the instructions of the vendors. The mouse CCL-5 ELISA kit was from Abcam (# ab100739), mouse CCR5 ELISA from Aviva Systems Biology (# OKEH 03441), mouse Arginase-1 ELISA from Abcam (# ab269541), mouse SDF-1 ELISA from Abcam (# ab100741), mouse Osteopontin ELISA from Abcam (# ab100734), mouse IL-6 ELISA kit from R&D (# M6000B), mouse IL-1 ELISA from R&D (# MLB 00C), mouse IL-10 ELISA from R&D (# M1000B), mouse IL-17 ELISA from R&D (# M1700), mouse IL-23 ELISA from R&D (# M2300), mouse TNFα ELISA from R&D (# MTA 00B) and the PGE2 ELISA from R&D (# KGE004B). Arginine was quantified using a commercial assay from abcam (# ab241028). All values were normalized to protein in the samples.Briefly, the tibias and femurs of the mice were isolated, the muscle removed and the ends of the bones were cut off. The bone marrow was collected by rinsing the shaft with 2-3 ml PBS per bone using a 25 g needle. The bone marrow suspension was aliquoted and shock-frozen in liquid nitrogen. Samples were thawed, sonicated 3-times for 10 seconds each using a tip sonicator (Ultrasonic GE50) to achieve complete lysis and homogenization of the samples. The aliquots were analyzed using ELISA for cytokines , arginine, PGE2.42, 0.7 mM MgCl2, 0.8 mM MgSO4), resuspended in 100 μL H/S, Fc-receptors were blocked by incubation for 30 min with Fc-block , samples were washed once and aliquots were stained with FITC-coupled anti-mouse CD45 antibodies  and APC-coupled antibodies against mouse CD4  or APC-coupled antibodies against mouse CD8  or PE-coupled antibodies against mouse CD19  or APC-coupled antibodies against mouse Ly6G (Gr1) . In addition, cells were stained with Alexa 647-coupled anti-mouse CD45 antibodies  and FITC-coupled antibodies against mouse Sca-1 . Cells were stained for 1 h at 4°C, washed once in H/S and analyzed on a FACS-Calibur. We analyzed the expression of the above described markers on 100 000 cells by flow cytometry.In order to determine cell numbers in the bone marrow, around 100.000 bone marrow cells were freshly isolated. Aliquots of bone marrows were washed once in H/S  in H/S plus 1% FCS for 45 min. Samples were washed again three times with PBS plus 0.05% Tween 20, once with PBS and were embedded in Mowiol. Samples were evaluated by confocal microscopy using a Leica TCS SL confocal fluorescence microscope.Samples were fixed in 4% paraformaldehyde (Sigma), buffered with PBS to pH 7.2 – 7.4, for 48 h and the bones were de-calcified by incubation in 25% (w/v) EDTA  for 4 days. The EDTA solution was changed daily. Samples were then de-hydrated in an ethanol to xylol gradient series, paraffin embedded, sectioned at 6 μm and dewaxed, rehydrated, and treated with pepsin  for 30 min. Sections were washed with water and PBS and blocked for 10 min at room temperature with PBS, and 5% fetal calf serum (FCS). The samples were then stained for arginase-1 employing a monoclonal mouse anti- murine arginase-1 antibody . Sections were incubated for 45 min in H/S at room temperature. Samples were then washed three times with PBS plus 0.05% Tween 20 and once with PBS and secondarily labeled with Cy3-coupled donkey anti-mouse F(ab)Immunostainings were controlled with isotype control antibodies that showed no or very weak staining. These were mouse IgG2a isotype control antibodies . We also included controls with secondary Cy3-coupled antibodies only.2.62 were determined by ELISA as above.Eight weeks old, male, C57BL/6 mice were intraperitoneally (i.p.) injected with linsitinib at 10 mg/kg twice daily for 2.5 days (60 hrs), thus, in total 5 times. Linsitinib was dissolved in DMSO at 25 mg/mL and diluted into 250 mM sodiumacetate (pH 5.0). Mice were sacrificed 4 h after the last injection, the bone marrow was removed and cells were isolated by flushing the bones to obtain bone marrow as above. Aliquots were directly used to determine arginase-1 expression and arginine concentrations as above. In addition, cells were washed once in H/S and resuspended in 5% fetal calf serum in H/S. Fetal calf serum does not contain measurable concentrations of arginine, which was confirmed by using the arginine kit as above. Cells were then incubated for 24 h in H/S plus 5% FCS without arginine or supplemented with 25 μM or 100 μM arginine. Cells were then shock-frozen and concentrations of IL-10 and PGE2.7Protein concentrations in the samples were measured using the BioRad Protein Assay Dye (#500006) and served to normalize the samples. If cells were suspended in 5% FCS in H/S, the protein content of FCS was also determined and subtracted.2.83H] arginine . Cells were then extensively washed 4-times in H/S and the remaining intracellular radioactive arginine was quantified by liquid scintillation counting.In order to determine the uptake of arginine into bone marrow cells, the cells were prepared as described above, resuspended in 5% fetal calf serum in H/S and incubated for 24 h with 10 μCi/mL [4,5-2.9The manuscript includes all data, no animals were excluded and no data was excluded. Thus the data follows the intent-to-treat principle, although it is certainly not a clinical study.p values for the pairwise comparisons were calculated after Bonferroni correction. P-values lower than 0.05 were considered as significant. In the ANOVA analysis we always compared the mean of each column with the mean of every other column and used the statistic program PRISM in order to calculate the adjusted p-values with the Bonferroni’s multiple comparison correction. The adjusted p-values are shown in the figures. The number of comparisons was 3 in all tests. The original p was set at 0.05, the corrected p is thus 0.05/3 = 0.0166. However, the computer program already adjusts the p-values.Statistical analyses were performed using Prism 7 (GraphPad Software) and accomplished with One‐Way‐ANOVA for multiple comparisons between the different groups applying the Bonferroni correction for multiple testing. The p value of 0.05 or lower (two-tailed). The sample size planning was based on the results of two-sided Wilcoxon-Mann-Whitney tests . Investigators were blinded to results of histologic analyses and to animal identity. Before the experiments, animals were randomly assigned to cages by a technician who was not involved in the experiments. Cages were randomly assigned to the various experimental groups. Data is shown as arithmetic mean ± standard deviation (SD), and the exact p-values are shown in the figures. Changes between mouse groups with p‐values > 0.05 are regarded as not statistically significant are not shown in the graphs.All values were normally distributed, and the variances were similar. Statistical significance was set at a 3In the present study, we investigated the role of bone marrow cells during development of experimental Graves’ disease and TED and whether the bone marrow is involved in the immune inhibitory effect of the IGF-1R/IR antagonist linsitinib. The study design is outlined in 3.1rd immunization . Immunized mice were left untreated or treated with linsitinib for 4 weeks starting 2 weeks after the 3nization . The bon3.2In order to further investigate stimulation of the bone marrow upon immunization with TSHR and the effects of linsitinib on this process, we determined the expression of Stem cell antigen-1 (Sca-1) by flow cytometry of freshly isolated bone marrow. Hematopoietic stem and progenitor cells (HSPCs) express the surface molecule Sca-1  and, thu3.3To compare the number of myeloid cells in the bone marrow between the different groups, mice were treated and bone marrow isolated as above. Bone marrow cells were stained with fluorescently-labelled anti-GR1 antibodies and analyzed by flow cytometry. GR1, also known as Ly-6G, is a cell surface marker that is primarily expressed on myeloid cells . Immuniz3.4In order to show the activation of the bone marrow via the CCL5(Rantes)-CCR5 axis, which has been previously implied as a major pathway activating bone marrow by T-lymphocytes during autoimmune encephalitis , we perf2 serve to down-regulate immune responses with potent anti-inflammatory characteristics   of linsitinib, twice daily. Control mice were injected with solvent. We then isolated bone marrow cells 4h after the last injection and determined arginase-1 expression and arginine concentrations in these samples. The results demonstrate that linsitinib induced expression of arginase-1 in the bone marrow (p ≤0.0001) (3.6in vitro. We then determined IL-10 and PGE2 concentrations. Linsitinib induced an increase of IL-10 and PGE2 concentrations in the bone marrow cells (p ≤0.0001) compared to untreated to cells in the medium lacking arginine, which mimics a high activity of arginase-1 with depletion of arginine in vivo  induces formation of myeloid-derived suppressor cells , 47. InhThus, linsitinib might upregulate arginase-1 expression by blocking important downstream signaling pathways of the IGF-1R, such as mTOR.Arginase-1 has been shown to be involved in the development of diabetes and it has been shown to be involved in insulin resistance by several mechanisms, including alterations of the balance between M1 and M2 macrophages and alteration of macrophage infiltration into fat tissue. It might be possible that arginase expression in the bone marrow induces a similar shift of macrophages to a phenotype corresponding to myeloid-derived suppressor cells and therefore contribute to immune suppression by linsitinib.Our studies suggest an important function of arginase-1 in the pathogenesis of GD and TED, a pathway which is targeted by linsitinib. Arginase-1 is a metalloenzyme that catalyzes the conversion of L-arginine to L-ornithine and thus is a key player in the urea cycle . The enz2 were down-regulated depending on arginase-1 inhibition, as evidenced in the reconstitution experiments, further escalating the autoimmune reaction. Linsitinib reversed these events by an upregulation of arginase-1 expression, depletion of arginine and upregulation/maintaining of IL-10 and PGE2, events that collectively prevent and inhibit the autoimmune response. IL-10, as an anti-inflammatory cytokine has also been shown to induce arginase-1 expression  to confirm the key findings of our studies obtained on immunized BABLB/c-mice in another mouse strain to exclude that the effects of linsitinib are restricted to a specific strain and (ii) we aimed to show that the mode of application (gavage vs i.p.) does not alter the effects of linsitinib on arginase expression.In addition, i.p. injection of linsitinib highly ensures that the drug (linsitinib) is absorbed, which is very useful as we also only injected the mice 5 times. On the contrary, this could not be performed with the initial linsitinib treatment via gavage, as i.p. injections for such a long time frame significantly increase the risk for infection.The studies confirmed that neither the mouse strain nor the application method alters the effects of linsitinib on arginase expression.Several studies have shown a crosstalk between the TSHR and IGF-1R, mainly in fibroblasts , 61, 62.Hyperglycemia is a potential adverse event of linsitinib, which we also investigated in our study by measuring the blood glucose levels of the mice . HoweverOur findings indicate that linsitinib leads to an upregulation of arginase-1, which decreases the availability of arginine for T-cells and thus inhibits the T-cell activation and inflammatory response. Thus, drugs that stimulate arginase-1 or increase expression of arginase-1 might be beneficial in thyroid eye disease and linsitinib is one example for such a novel drug. Moreover, treatment with arginase-1 or arginase-1 activators/inducers might potentiate the effect of linsitinib, making it an even more efficient drug. However, it is important to find the right balance between the immunosuppressive effect on the bone marrow, which is wanted and needed in order to treat autoimmune disorders, and complete immunosuppression allowing, for instance infections.In addition, arginase-1 could serve as a prognostic biomarker. Patients with high levels of arginase-1 in the blood or bone marrow should have a milder course of the disease then patients with low levels of arginase-1. However, these are just speculations which need to be tested in clinical studies.The data are the first report of a role of the bone marrow in the pathophysiology of TED. However, they have not been confirmed by performing the same experiments on an 2nd independent mouse cohort and, therefore, should be considered in this respect as preliminary until independent confirmation. The method of immunization and induction of the autoimmune condition Graves’ Disease has been independently repeated by several studies from different scientist, also several times from our lab in which we identified the mouse model as robust and reproducible , 63–65.5In summary, we investigated the development of Graves’ Disease and thyroid eye disease in a murine mouse model after immunization with the A-subunit of the TSHR. We suggest the bone marrow as a new key player to trigger the autoinflammatory response of the disease and to promote disease development of thyroid eye disease. The oral, small molecule IGF-1R and IR antagonist linsitinib blocks the activation of the bone marrow and thus inhibits disease progression and development of TED. Linsitinib reduces proinflammatory cytokines and inflammation in the bone marrow and inhibits T-cell activation by upregulation of the enzyme arginase-1. This local immunosuppressive effect of linsitinib and the potential importance of the bone marrow in TED development gives insight into a new understanding of the disease and potential new therapeutic options to treat TED which implicates the clinical significance of our findings.The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author.The animal study was approved by the North Rhine Westphalian State Agency for Nature, Environment and Consumer Protection (LANUV), Germany, or by the local IACUC, University of Cincinnati, Cincinnati, USA. All experiments were performed according to the FELASA regulations and ARRIVE guidelines. The study was conducted in accordance with the local legislation and institutional requirements.AG: Study design, data collection and analysis, performing of statistical analysis, writing and preparing of the original draft, project administration; MH: helped with data collection, SK: helped with the ELISA, MS: performed bone marrow sections, BW: helped with the ELISA, GW: Resources, Reviewing of the manuscript, RZ: Funding Source, resources, GH: Resources, AD: helped with data collection, NB: Resources, AE: Resources, supervision, Reviewing and Editing of the manuscript, G-EG: helped with the data collection and analysis, resources, supervision, reviewing and Editing of the manuscript.


The results showed that the addition of pectin could overcome the phenomenon of high-MTGase-induced lower gelling strength of gelatin gels. At a low pectin content, the modification sequences had non-significant influence on the gelling properties of modified FG, but at a higher pectin content , P0.5%-FG-TG had higher gel strength (751.99 ± 10.9 g) and hardness (14.91 ± 0.33 N) values than those of TG-FG-P0.5% . Rheology analysis showed that the addition of pectin normally improved the gelation points and melting points of FG. The structural results showed that the fluorescence intensity of FG was decreased with the increase in pectin concentration. Fourier transform infrared spectroscopy analysis indicated that the MTGase and pectin complex modifications could influence the secondary structure of FG, but the influenced mechanisms were different. FG was firstly modified by MTGase, and then pectin (P-FG-TG) had the higher gelling and stability properties.In this paper, the effects of different modification orders of microbial transglutaminase (MTGase) and contents of pectin (0.1–0.5%,  Gelatin is a class of biopolymers which is widely used in modern manufacturing industries, especially in food processing, pharmaceutics, and cosmetics, due to its unique functional properties  . GelatinCompared with mammalian gelatin, fish gelatin (FG) has lower proline and hydroxyproline contents, and these amino acids are responsible for the stability of collagen like the triple helix structure of gelatin. Thus, FG has lower gel strength and gelling and melting temperatures, which limit its application . In ordeTherefore, this study mainly investigates the orders of MTGase and pectin complex modifications on the gelling and rheological properties of FG through gel strength, texture profile analysis (TPA), and rheological analysis. Moreover, the modification mechanisms were also discussed through the analysis of endogenous fluorescence, FTIR, and gels structure. We hoped this paper would mainly provide an important theoretical basis for the application of complex modifications for the improvement of the functional properties of gelatin.Tilapia skin gelatin  was purchased from the Yuanye Company . Microbial transglutaminase  was obtained from Yiming Company . High-methoxyl citrus pectin  was obtained from Aladdin . All other chemical reagents are analytical grade.w/v, pH 6.5) was prepared through dissolving FG in non-ionic water at 40 °C. Then, the orders of modification FG by MTGase (0.005%), various contents of pectin  were divided into two groups: TG-FG-P and P-FG-TG. TG-FG-P: Firstly, different concentrations of pectin were added to FG solution and stirred at 40 °C until the pectin was completely dissolved. Then, MTGase was added into the mixture solution and cross-linked at 40 °C for 40 min. The enzyme was inactivated at 100 °C for 5 min. P-FG-TG: MTGase was firstly used to modify FG, and the mixture was incubated at 40 °C for 40 min for the cross-linking. And then, the enzyme was inactivated at 100 °C for 5 min. Subsequently, different concentrations of pectin were added into the mixture and stirred at 40 °C until completely dissolved to form stable composite colloid. After reaction, all the gelatin solution was finally cooled down to form a stable composite colloid and stored at 4 °C for further use.Fish gelatin (FG) solution (6.67% w/v) was transferred into a 25 mL glass beaker, and then put into a refrigerator at 4 °C for 16–18 h to form the gels. The gel strength was measured using a texture analyzer  with a probe of P 0.5R . The gel strength was recorded at a speed of 1.00 mm/s when the probe penetrated the gel for 4 mm. Each sample was performed in triplicates.According to the method of Sha et al.  with minw/v) was put into a cylindrical container and matured in a refrigerator at 4 °C for 16–18 h. The gels were subjected to two cycle compressions to 40% of their original height at a speed of 1 mm/s with a P 36R probe . The deformation of gel was 40%, and each sample was tested three times. Finally, the hardness, elasticity, cohesion, gelation, and chewiness of the colloid were obtained [TPA of gelatin gels was performed using a TA.XT plus texture analyzer  equipped with the Peltier temperature control system (4 °C). Gelatin solution  were measured using a controlled rheometer  with a stainless-steel parallel plate (60 mm diameter). Silicone oil was smeared evenly around the parallel plate before experiment. The gap between the fixture and base was 1000 μm. The FG solution was first cooled from 40 °C to 4 °C, kept at 4 °C for 5 min, and then heated from 4 °C to 40 °C, with a rate of 0.5 °C/min, a strain force of 0.5%, and at a frequency of 1 Hz. The gelation point (Gp) and melting point (Mp) could be obtained during the cooling and heating procedure, respectively [The storage modulus (G′) and loss modulus (G″) of all gelatin solution .The transparency of all gelatin solutions was determined with an ultraviolet visible spectrophotometer at a wavelength of 600 nm ,16. DistA fluorescence spectrophotometer  was used to measure the fluorescence intensity for all gelatin solution . The 2 m−1 at 25 °C. The obtained automatic signals were collected in 32 scans with a resolution of 4 cm−1, and each sample was subjected to three parallel tests.The FTIR analysis of samples was performed using an FTIR spectrometer  . The lyoAccording to the method of María S et al. , the mic0.1% gel displays the highest gel strength and hardness values. The addition of a certain amount of pectin can form a stable FG-P polymer mixture through electrostatic interaction [0.5%-FG-TG has the highest values. MTGase firstly catalyzes lysine and glutamine in FG to form super polymers, and the addition of pectin still could interact with super-FG molecules through electrostatic interactions to form macro-molecular polymers, enhancing the hydrogen bonds of the gelatin gels and increasing the gel strength and hardness values of FG-TG composite colloids with the increase in pectin concentration. The gumminess and chewiness of all composite colloids almost show a similar trend of gel strength and hardness. Interestingly, the three modification methods have non-significant differences on the springiness, cohesiveness, and resilience of all the gelatin gels. Similarity, Huang et al. [Gel strength is one of the main physical properties of gelatin, and it is a stiffness factor that can predict the quality of gelatin. TPA can better simulate the effect of the tongue and teeth on gel . As showeraction . Followieraction . For theg et al. ,10 also Gp) and melting point (Mp) [01%-FG-TG is also higher than that of P0.5%-FG-TG. This shows that the molecular arrangement of FG modified by MTGase and then added with low-concentration pectin is denser with better viscoelasticity and higher molecular stability than other samples. The dynamic viscoelastic behavior (DVB) of gelatin samples in the temperature range of 40~4 °C and 4~40 °C was monitored to obtain the gelling point (int (Mp) . As showint (Mp) ,21. At 4Gp and Mp [Gp and Mp of the gelatin samples slightly increased, and all the complex modified gelatin gels had higher Gp and Mp values than those of pure FG. However, the modification orders of MTGase and pectin have non-significant influence on the Gp and Mp. For the TG-FG-P group, FG interacts with pectin to form a super-polymer; MTGase catalyzes this super-polymer or cross-links with gelatin to form an interconnected molecular ring structure, thus improving the Gp and Mp. For the P-FG-TG group, MTGase catalyzed FG molecules to form double bonds; after the deactivation of MTGase, pectin and FG were polymerized through hydrogen bonding to form super-polymers, thus improving the Gp and Mp of gelatin. Nevertheless, the TG-FG-P group and P-FG-TG group had higher Gp and Mp values than those of FG-TG, especially the complexes with high pectin contents. This suggested that the MTGase and pectin complex modification had a higher improvement efficiency than that of pure MTGase modification.The crossover points of G′ and G″ on the cooling and heating curves are defined as the p and Mp ,23. It cTurbidity represents the formation of insoluble compounds. The size of turbidity is not only related to the content of colloidal substances in water, but also to the size and shape of these substances, usually closely related to the stability of protein polysaccharide systems . The hig0.3%. Studies have shown that pectin has a quenching effect on the fluorescence groups of gelatin [Tyrosine and phenylalanine in gelatin are the main fluorescent substances, and fluorescence analysis can reflect the minor changes in the tertiary structure of protein ,27. Acco gelatin . When FG2 residues, which is related to the formation of intra-molecular hydrogen bonds. As shown in 2, leading to the reduction of amide A. Normally, the amide A of P-FG-TG is lower than that of TG-FG-P. This because in P-FG-TG, MTGase firstly catalyzes the cross-linking of FG to form covalent bonds, reducing the amino acids that can form hydrogen bonds, resulting in fewer hydrogen bonds formed when pectin interacts with FG. In the TG-FG-P group, pectin interacts with FG to form a stable complex via the formation of hydrogen bonds. At this time, fewer catalysis sites could be used to form covalent bonds among FG molecules with the influence of MTGase. Thus, the TG-FG-P group had less covalent bonding. This might explain why P-FG-TG had higher gel strength and hardness than those of the TG-FG-P group. Amide I mainly exhibits C=O stretching, while amide II mainly exhibits N-H deformation [2 of gelatin and C=O of pectin, which was due to the increase in inter-molecular hydrogen bonds. Thus, both complex modification orders could improve the gelling properties of TG-modified FG.FTIR can be used to analyze the changes in functional group information attached to protein skeletons, reflecting changes in the secondary structure of proteins . Amide Aormation . The ami0.3% has the densest network with smaller numbers of pores. Low-concentration pectin can be trapped in the original pores through the formation of inter-molecular hydrogen bonds, reducing the catalytic sites of MTGase, forming FG-P and FG-FG network structures [In order to better observe the modification orders on the microstructure of modified gelatin gels, SEM was used to analyze the sub-micron gel network structure . As showructures ,10. Howeructures , improviIn conclusion, the gel network of complex modified gelatin is the most uniform and fine, which indicates that the complex modification can overcome the phenomenon “excessive MTGase weaken gelling properties of FGG″.In order to study the effects of different modification orders of MTGase and pectin on the gelling properties and molecular structure of FG, the modification mechanisms were established. As shown in Gp and Mp values) than those of pure FG and FG-TG. At the same pectin content, compared with the TG-FG-P group, the P-FG-TG group had slightly higher gelling properties and stability, and P0.5%-FG-TG had the highest gelling properties. Structural results showed that complex modified gelatin gels had the densest gel network compared to both FG and TG-FG, but the TG-FG-P group had fewer covalent bonds than the P-FG-TG group. Through the viewpoints of gelling properties, MTGase-pectin modification (P-FG-TG) could be used to produce gelatin with high quality. However, further studies still should be performed to evaluate its true application value in the food, e.g., yoghurt, and release delivery systems.Herein, the different complex modification orders of MTGase and pectin on the gelling and structural properties of FG were comparatively investigated. The results showed that two kinds of complex modification orders could overcome the drawback of high MTGase causing gelling weakness, and almost all complex modified gelatin gels had higher gelling properties (gel strength, hardness,


Background: Spontaneous bacterial peritonitis (SBP) is a common infection in patients with cirrhosis and ascites. Currently, the accuracy of the model for end-stage liver disease (MELD) and MELD-sodium (MELD-Na) as prognostic scores in this cohort is unclear. This study aimed to evaluate and compare the accuracy of MELD and MELD-Na for predicting 90-day mortality and determine whether the mortality risk estimates they provide accurately reflect the poor prognosis of patients with SBPMethods: Patients with cirrhosis and SBP were retrospectively identified from ascitic fluid samples sent for microscopy, culture and sensitivity analysis (1/1/18-31/12/20) and a previous audit. MELD and MELD-Na scores at diagnosis were calculated and associations with 90-day mortality were assessed using univariate analysis. Receiver operator characteristic curves were compared, and standardised mortality ratios (SMRs) were calculated by comparing the number of deaths observed to the number predicted by MELD and MELD-Na.Results: Of the 567 patients identified, 15 patients with cirrhosis and SBP were included. The 90-day mortality rate was 66.7% (10/15). Only concurrent hyponatremia (<135mmol/L) was associated with mortality . The difference in MELD and MELD-Na’s C-statistic was not significant: 0.66 (95% Cl:0.35-0.98) vs 0.74 (95% Cl:0.47-1.0) respectively (p=0.72). Patients with a MELD-Na >18.5 had significantly higher 90-day mortality than patients with MELD-Na ≤18.5 (88.9% (8/9) vs. 33.3% (2/6), p=0.05). The SMR (95% Cl) for each MELD decile evaluated was 33.3 (0-79.5), 11.1 (0.2-22.0) and 3.4 (0-7.0) for scores ≤9,10-19 and 20-29 respectively. For each MELD-Na tertile, these were: 25 (0-59.6), 5.2 (0.1-10.3) and 2.7 (0.1-8.1) for scores <17,17-26, ≥27 respectively.Conclusion: In a small cohort of patients with cirrhosis and SBP, the MELD’s accuracy in predicting 90-day mortality was limited. MELD-Na’s accuracy was higher but not significantly. Both scores consistently underestimated participants’ mortality, therefore future studies could evaluate the accuracy of alternative prognostic scores in this patient group. Spontaneous bacterial peritonitis (SBP) is defined as a bacterial infection of the ascitic fluid that occurs in the absence of any surgically treatable intra-abdominal sources of infection . SBP is The model for end-stage liver disease (MELD) is a widely used prognostic score in patients with cirrhosis. Originally designed to predict short-term survival following elective transjugular intrahepatic portosystemic shunt (TIPS) placement , it subsOutside of the context of donor liver allocation, MELD has also been validated as a prognostic score in multiple other cohorts: for example, to predict short-term mortality in patients with cirrhosis undergoing elective and emergency surgery  and in aAt present, few studies have formally evaluated the predictive accuracy of MELD in patients with cirrhosis and SBP and the results so far have been conflicting ,20,21 boSecondly, it is unclear whether the mortality risk estimates associated with each decile of the MELD score accurately represent the high short-term mortality of patients with SBP . PreviouA commonly utilised modification of MELD is the model for end-stage liver disease sodium (MELD-Na). Hyponatremia, which develops in patients with cirrhosis due to excess fluid retention disproportionate to that of sodium  has beenThis retrospective study aims to evaluate and compare the predictive accuracy of MELD and MELD-Na for predicting 90-day mortality in patients with cirrhosis and SBP. In addition, by comparing the mortality of study participants to the mortality-risk estimates associated with each MELD  and MELDStudy designThis was a retrospective study conducted as part of a local quality improvement project at Northwick Park Hospital (approved in April 2021). No formal ethical approval was required, and no patient-identifiable information was collected. Patient management was not affected.PatientsTo identify eligible patients for this study, all consecutive ascitic fluid samples sent for microscopy, culture and sensitivity analysis at London Northwest University Healthcare NHS Trust  between 1st January 2018 and 31st December 2020 were considered retrospectively. In addition, patients diagnosed with SBP between 1st May 2019 and 30th April 2020 at Imperial College Healthcare NHS Trust  who had been identified prior to the start of the present study for audit purposes were considered for eligibility.The inclusion criteria were adults (≥18 years of age) with cirrhosis and an ascitic fluid neutrophil count >250 cells/mm3 (culture-positive or negative) . The preThe exclusion criteria were any evidence of a surgically treatable intra-abdominal source of infection , active For patients who experienced multiple admissions with SBP during the time periods included, only the first admission was included.Data collectionPatients’ demographic and clinical information at presentation including age, sex, ethnicity and aetiology of cirrhosis was obtained from electronic clinical records. The presence of concurrent acute kidney injury (AKI), upper gastrointestinal haemorrhage and hepatic encephalopathy on presentation with SBP was noted from electronic patient notes and discharge summaries. Hyponatremia was defined as a serum sodium concentration of <135mmol/L33 and was noted using laboratory findings at the time of abdominal paracentesis.The study outcome was all-cause mortality at 90 days from the date of diagnostic paracentesis which was also noted from electronic patient records.MELD and MELD-Na scoresPrognostic scores were calculated from laboratory tests that occurred within the 24 hours directly preceding or following diagnostic paracentesis. Where multiple tests had been completed within this timeframe, the test performed closest in time to that of the paracentesis was used to calculate a patient’s score.MELD scores were calculated using an online calculator  with theMELD-Na scores were calculated using an online calculator  with theStatistical analysisContinuous variables were assessed for normality using the Shapiro-Wilk test. Results are presented as means and standard deviations for continuous parametric data, medians and interquartile ranges for continuous non-parametric data and frequencies and percentages for categorical data. Univariate analysis was performed to assess for associations with 90-day mortality using unpaired, two-tailed t-test, Mann-Whitney U test and Fischer’s exact test for each data type respectively.ROC curves for each model were plotted and the accuracy of MELD and MELD-Na to predict 90-day mortality was assessed using concordance (c)-statistics . C-statistic values range from 0-1; higher values indicate that a model can rank patients according to their outcomes more accurately. A c-statistic greater than 0.7 suggests a model is clinically useful; a score between 0.8-0.9 indicates excellent discriminatory ability . C-statiThe specificity and sensitivity of each MELD and MELD-Na cut-off were assessed visually for that with the highest values to determine if a specific value of MELD and MELD-Na could act as a threshold for potential risk-stratification. Kaplan-Meier survival curves for patients above and below the cut-off chosen were compared using the log-rank test.To evaluate whether MELD and MELD-Na underestimated the mortality of study participants, standardised mortality ratios (SMRs) were calculated. Patients were stratified by MELD score into deciles  and by MELD-Na score into tertiles . In each grouping, the number of deaths expected according to MELD and MELD-Na was calculated using the 90-day mortality rates published by Wiesner et al.  and Kim All analyses were conducted using GraphPad Prism v9.1  except for the calculation of SMRs where Microsoft Excel (v16.49) was used. For all tests, a p-value of ≤0.05 was considered significant except for SMRs, where a 95% confidence interval that did not cross one was considered significant.Across the two NHS trusts, 567 patients in total were considered for eligibility. As shown in Figure Patients and outcomeThe demographic and clinical characteristics of the study cohort and laboratory findings at diagnosis with SBP are shown in Table Of the 15 patients included, alcoholic liver disease (ALD) was the most common cause of underlying cirrhosis, affecting 10 patients. Hepatitis C and the combination of non-alcoholic fatty liver disease (NAFLD) and ALD were each responsible for cirrhosis in two patients. In the remaining patient, primary biliary cholangitis (PBC) was the cause of cirrhosis.Alongside SBP, five patients also presented with an AKI and five with hyponatremia. One patient presented with both additional features concurrently. Five out of the 15 patients had hepatic encephalopathy at diagnosis, and one patient also presented with an upper gastrointestinal haemorrhage. Of the five patients with culture-positive SBP, the organism was identified as Escherichia coli in four cases and as Klebsiella pneumoniae in the remaining patient.The mean MELD score at the time of diagnosis was 18.9 ±7.3 and the mean MELD-Na score was 20.8 ±6.9. At 90 days, 10 patients out of 15 patients included had died (66.7%). The median survival time was 45 days.Factors associated with 90-day mortalityAs shown in Table Other variables that were close to reaching statistical significance included MELD-Na score and white blood cell (WBC) count, both of which were higher in the non-survivors; 21.8 ±6.8 compared to 15.4 ±6.4 (p=0.10) and 14.7 ±7.9 x109/L compared to 6.2 ±4.8 x109/L (p=0.07) respectively. Serum sodium and albumin were both lower in non-survivors: 132.7 ±7.6 mmol/L compared to 139.2 ±1.8 mmol/L (p=0.09) and 26.4 ±7.1 g/L compared to 31.8 ±5.9 g/L (p=0.18) respectively.MELD score was higher in patients who died compared to patients who survived; 17.8 ±6.5 compared to 14.4 ±6.5 (p=0.36). Of the three components of MELD, creatinine was also higher in non-survivors; 118.0 (66.8-300.8) µmol/L compared to 90.0 (65.5-124.0) µmol/L (p=0.31). In contrast, bilirubin and INR were almost identical between the two groups: bilirubin was 30.5 (7.8-87.5) µmol/L in non-survivors compared to 29.0 (17.0-47.0) µmol/L in survivors (p=0.70), whilst INR was 1.4 ±0.4 compared to 1.5 ±0.3 in non-survivors and survivors respectively (p=0.41).Predictive accuracy of MELD and MELD-NaFigure The MELD score with the highest sensitivity and specificity to predict 90-day mortality was a cut-off of >16.5, with values of 0.7 and 0.8 respectively. Figure The optimum cut-off for MELD-Na to predict 90-day mortality was a score of >18.5; sensitivity and specificity were both 0.8. As shown in Figure Accuracy of MELD and MELD-Na’s mortality risk estimatesFor each decile of the MELD score evaluated, the SMR was greater than one as the number of deaths observed was higher than the number predicted by MELD Table . As the For all MELD deciles analysed the observed number of deaths was not significant compared to the number predicted by MELD. As no patients had a MELD score greater than 29, the calibration of MELD in patients with SBP and MELD scores of 30-39 or ≥40 was not evaluated.For patients of all MELD-Na tertiles , the number of deaths observed was higher than the number predicted Table . As withSBP is a common infection in patients with cirrhosis and ascites and confers high short-term mortality. At present, the accuracy of the widely used prognostic scores MELD and MELD-Na in this cohort is unclear. By evaluating these scores’ ability to predict 90-day mortality and whether the risk estimates they provide are accurate in patients with SBP, this study adds to our understanding of the areas of practice where MELD and MELD-Na may be useful tools for clinicians and researchers to utilise in this patient group and where a more cautious interpretation may be required.In this study, MELD’s c-statistic of 0.66 fell below the generally accepted cut-off of 0.7 to consider a prognostic score clinically useful . This liIn another study by Nobre et al. , bilirubThe addition of serum sodium into MELD to produce MELD-Na showed a non-significant improvement in the model’s discriminatory ability despite hyponatremia’s observed association with 90-day mortality. The lack of superiority using MELD-Na appears to conflict with previous studies where MELD-Na but not MELD was an independent predictor of short-term mortality in this cohort . PreviouThe optimum cut-off for MELD-Na (>18.5) but not for MELD (>16.5) was able to stratify patients into two groups with significantly different 90-day mortality rates. However, when considering if this stratification could be useful in clinical practice, it should be noted that the 90-day mortality of patients with MELD-Na scores below or equal to this cut-off was not inconsiderable (33.3%). Therefore, to classify this group as “low” risk and in need of less intensive monitoring or treatment appears inappropriate. In a larger study by de Oliveria Coberllini Jacques et al. , althougBoth MELD and MELD-Na underestimated the 90-day mortality of patients in this study, although no differences in the number of observed and predicted deaths at each decile or tertile respectively reached statistical significance. As discussed previously, this result should be considered with the study’s sample size in mind, as larger studies have reported MELD to underestimate mortality in specific SBP cohorts and where patients with cirrhosis and bacterial infections more generally were included ,47. To oFrom these results, we can only hypothesise why these scores do not appear to accurately represent this cohort’s poor prognosis. In univariate analysis, WBC count was almost significantly higher in non-survivors compared to survivors and in larger studies; this has consistently been reported as an independent predictor of mortality in this cohort ,48. WhilWhilst inflammation and its clinical consequences appear to have an important prognostic role in this cohort, MELD and MELD-Na do not include either direct measures of inflammation in their calculation, such as WBC count or c-reactive protein, or account for the extra-hepatic dysfunction as part of ACLF, as they do not include variables which reflect the function of the many different organ systems that may be affected. In ACLF cohorts, the CLIF-Consortium ACLF (CLIF-C ACLF) prognostic score which considers a patient’s circulatory, respiratory, and cerebral function in addition to renal, hepatic and coagulation dysfunction represented by MELD showed a significantly improved ability to predict 90-day mortality .Outside of the context of ACLF, systemic inflammation in SBP is associated with other clinical consequences which these scores may not account for. Higher levels of pro-inflammatory cytokines on diagnosis have been associated with increased haemodynamic dysfunction and are suggested as a key factor in the pathogenesis of HRS development ,52. PrevA particularly interesting consideration is whether these models may underestimate the mortality of all patients with SBP or if only certain groups are affected. In this study, the magnitude of underestimation was non-uniform across different MELD and MELD-Na score categories, with the largest difference between actual and predicted mortality observed in patients with the lowest prognostic scores (MELD ≤9 and MELD-Na <17). Kimmann et al.  reportedWhen considering this study’s findings, one should also be mindful of its limitations. Firstly, the prevalence of SBP in the time periods analysed was considerably lower than reported previously ,3. This As the electronic records used to collect information for each study participant do not include prescription charts, no information was obtained on the treatment and interventions that the study cohort received. We assumed patients were treated in line with current EASL guidelines , howeverDue to the study’s retrospective nature, the laboratory tests used to calculate MELD and MELD-Na scores were dependent on when these had been undertaken during a patient’s admission. Whilst all scores were calculated using tests performed within 24 hours of diagnostic paracentesis, this could have resulted in scores used which do not in fact represent a patient’s true MELD or MELD-Na score on diagnosis with SBP.Alongside addressing these limitations, a key consideration for future work is to evaluate and characterise any real-world consequences that may affect this patient group due to potential inaccuracies in these scores. Patients with end-stage liver disease have described confusion over their disease trajectory previously  althoughAnother aspect to consider is whether patients with SBP are disadvantaged in referral to liver transplant assessment units or in the allocation of donor livers. As a UK-based study, we did not assess this as UKELD  is utiliGiven its apparent prognostic role in this cohort, the predictive accuracy and calibration of models which include measures of systemic inflammation should be evaluated as potential alternatives. One such example is the CLIF-Consortium Acute Decompensation score (CLIF-C ADs) which incorporates WBC count  . In the In conclusion, in this small cohort of patients with cirrhosis and SBP, MELD had limited ability to predict 90-day mortality. Whilst hyponatremia was associated with mortality, the inclusion of serum sodium concentration in MELD-Na showed a non-significant improvement in predictive ability compared to MELD alone. The risk estimates provided by both models appeared to underestimate study participants’ 90-day mortality, therefore, alongside validating the present findings, future studies should consider evaluating the calibration of alternative prognostic scores in this patient group.


Chemotherapy with immune checkpoint inhibitors is the new standard of care for first-line systemic therapy in extensive small-cell lung cancer. The identification of biomarkers for patients that are likely or not likely to respond to such therapy is critical. We aimed at determining whether imaging could help to predict outcomes among these patients. We showed that the total metabolic tumor burden, extracted from pre-treatment 18-FDG PET/CT imaging, may be a useful biomarker associated with survival. Finally, we think that this result should be taken into account in clinical trials, and that it might need further validation through large, independent, and prospective cohorts.Background: We aimed to evaluate the prognostic value of imaging biomarkers on 18F-FDG PET/CT in extensive-stage small-cell lung cancer (ES-SCLC) patients undergoing first-line chemo-immunotherapy. Methods: In this multicenter and retrospective study, we considered two cohorts, depending on the type of first-line therapy: chemo-immunotherapy (CIT) versus chemotherapy alone (CT). All patients underwent baseline 18-FDG PET/CT before therapy between June 2016 and September 2021. We evaluated clinical, biological, and PET parameters, and used cutoffs from previously published studies or predictiveness curves to assess the association with progression-free survival (PFS) or overall survival (OS) with Cox prediction models. Results: Sixty-eight patients were included (CIT: CT) (36: 32 patients). The median PFS was 5.9:6.5 months, while the median OS was 12.1:9.8 months. dNLR (the derived neutrophils/(leucocytes-neutrophils) ratio) was an independent predictor of short PFS and OS in the two cohorts (p < 0.05). High total metabolic tumor volume (TMTVhigh if > 241 cm3) correlated with outcomes, but only in the CIT cohort . Conclusion: Baseline 18F-FDG PET/CT using TMTV could help to predict worse outcomes for ES-SCLC patients undergoing first-line CIT. This suggests that baseline TMTV may be used to identify patients that are unlikely to benefit from CIT. At diagnosis, on average, two-thirds of patients with small-cell lung cancer (SCLC) have extensive-stage (ES) disease. The prognosis is very poor, with a 5-year survival rate of 7% . DespiteOnly a few ES-SCLC patients experience a long-term benefit under ICI, and it is much more difficult to predict this benefit at an individual level in comparison with metastatic non-small cell lung cancer patients. To date, there are no useful predictive biomarkers to select the subgroup of patients that might benefit from immunotherapy in the long term. Therefore, the identification of biomarkers for patients that are likely to respond to ICI therapy is critical, and might be a relevant step in enhancing the standard of care for ES-SCLC patients .In the era of immunotherapy and personalized medicine,18F-FDG PET/CT is a functional molecular imaging modality used to assess several aspects of the disease status of oncological patients, including disease staging, prognosis, and response to therapy . Among tRecent evidence has also suggested that a high TB harms anticancer immunity; hence, a high metabolic tumor volume in patients treated with ICI was also shown as a significant prognostic biomarker in some cancers ,10,11,12However, to our knowledge, the prognostic value of baseline metabolic tumor burden on 18F-FDG PET/CT has never been evaluated yet in patients with ES-SCLC treated using chemo-immunotherapy.The present study aimed to determine if 18F-FDG PET/CT parameters can predict outcomes in patients with ES-SCLC undergoing first-line CIT. To assess the possible prognostic or predictive impact on the outcomes, we also included a control group of patients treated using chemotherapy alone.We carried out a retrospective study across multiple centers of patients with ES-SCLC who were treated with either chemotherapy alone or a combination of chemotherapy and first-line immune checkpoint inhibitors (ICIs). These patients underwent 18-FDG PET/CT scans at baseline, between June 2016 and September 2021, as part of standard care in three French centers. From May 2019, patients could receive immunotherapy from an early-access program in ES-SCLC. For the CT group, we analyzed a historical cohort of consecutive patients treated before this early-access program.The flow-chart is provided in Patients were eligible if they had: (i) biopsy-proven SCLC, (ii) stage IV disease, or (iii) treatment with platinum-etoposide chemotherapy alone (CT group) or in association with first-line PD-L1 inhibitors, atezolizumab, or durvalumab (CIT group). Patients were excluded if: (i) the delay between FDG PET/CT and the first ICI perfusion was >7 weeks (n = 7), (ii) the follow-up was <6 months, defined as the delay between the last contact and the diagnostic (n = 7), or (iii) they had another primary malignancy (n = 10). The inclusion criteria for blood samples assessment before ICI and chemotherapy was 7 days. Demographic, clinical, pathological, biological, and molecular data were also collected.After a fasting time of at least 6 h, 18F-FDG PET/CT scans were performed 60 min  after the injection of 18F-FDG . In most cases, images were obtained from the skull vertex to the proximal femur. Images were acquired and reconstructed according to current guidelines  using siAll hypermetabolic metastatic lesions were selected for the analysis, while hypermetabolic foci explained by inflammatory or physiologic activity were excluded. As a measure of the tumor glycolytic activity, the 18F-FDG PET/CT uptake was quantified by the maximum standardized uptake values normalized by body weight (SUVmax). To assess the metabolic tumor burden, MTV was measured by setting a margin threshold of 42% of SUVmax . The tumThe primary endpoint analysis was to build a multivariable model using pretreatment 18F-FDG PET imaging to predict the outcome. Progression-free survival (PFS) was defined as the delay between the first CIT or the CT perfusion, and the disease progression or the death from any cause; and overall survival (OS) was defined as the delay between the first ICI-chemotherapy or chemotherapy administration, and the death from any cause or censoring at the time when the patient was known to be alive. Follow-up was determined from the first ICI-chemotherapy or chemotherapy perfusion to the date of the last clinical consultation. The assessment of outcome was blinded.p-value of less than 0.05 was deemed statistically significant. Analyses were performed with RStudio .For the derived neutrophils/(leucocytes–neutrophils) ratio (dNLR), we used the cutoff of 3 (>3 vs. ≤3) from the largest published study with ICIs . For bio3 vs. ≤241 and Tumor SUVmax > 12 vs. ≤ 12 (vs. ≤ 12 .p > 0.05). Furthermore, there was no significant relationship between these biomarkers, and clinical or biological parameters (Spearman’s correlation coefficient < 0.3). As an example, when testing the relationship between TMTV and dNLR, Spearman’s correlation coefficient was 0.08 (p > 0.10).Correlations between PET biomarkers, extracted from tumor lesions and variables of interest, are presented in The median PFS was 5.9 (95% CI 5.2–7.9) months in the CIT group and 6.5 (95% CI 4.6–7.6) months in the CT group. In the study, progressive disease was observed in 96% of the patients (65 patients), with 34 patients (94%) from the CIT group and 31 (97%) from the CT group. The AUCs for the predictions of PFS at 6, 12, and 18 months in the two groups based on TMTV and dNLR are provided in After conducting a multivariable analysis, the study found that both a high dNLR and a high TMTV were independent and statistically significant prognostic factors in the CIT group . However, only a high dNLR was found to be statistically significant in the CT group .p > 0.05 for both; TMTV and dNLR did not provide any additional prognostic value to the multivariable model obtained with backward elimination for PFS in the CIT group (The study’s results showed that the median OS was 12.1 months (95% CI 9.6–17.0) in the CIT group, and 9.8 months (95% CI 7.5–15.3) in the CT group. Of the total of 68 patients, 56 patients  died, with 25 (69%) in the CIT group and 31 (97%) in the CT group. The AUCs for the prediction of OS at 6, 12, and 18 months in the two groups based on TMTV and dNLR are provided in p = 0.06). High Tumor SUVmax, brain metastases, age > 70 years, and PS ≥ 2 did not correlate with OS in the two groups.The univariable analysis revealed that high dNLR and liver metastases (LM+) were both associated with poor OS in the CIT group . HoweverAccording to the analysis of multiple variables, having a high dNLR was found to be a significant and independent factor for predicting outcomes in both groups . The presence of liver metastases did not remain as a significant predictor for OS in the CIT group .p < 0.05 for both; Furthermore, TMTV and dNLR added significant prognostic values to the multivariable model obtained with the backward elimination process for OS in the CIT group , intermediate- (TMTVhigh or LM+ n = 10 in the CIT cohort and n = 10 in the CT cohort), and high risk (TMTVhigh and LM+ n = 8 in the CIT cohort and n = 7 in the CT cohort).We combined the TMTV and LM status , both with p < 0.01: the statistical difference between the high-risk group versus the intermediate/low-risk groups)  for the high-risk group versus 13.9 months (95%CI 11.6-NA) for the intermediate risk group versus 14.1 months (95%CI 12.0-NA) for the low-risk group ( groups) . At 12 m groups) . This TM groups) . In the  groups) .This study explored the prognostic values of specific metabolic parameters from the pretreatment 18F-FDG PET/CT of ES-SCLC patients undergoing first-line therapy with CIT or CT. The prognosis of patients displaying a high metabolic TB before first-line CIT was worse than patients displaying a low metabolic TB. A similar trend was observed for the OS analysis in the CIT group; however, TMTV did not reach statistical significance.3), using predictiveness curves, which is consistent with other studies (a cut-off at 245.7 cm3 was used in a recent study for a patient treated with first-line treatment in ES-SCLC [We identified a relevant cut-off to determine patients displaying a high versus low TMTV (> or ≤241 cm ES-SCLC ). A rece ES-SCLC  showed tRecent evidence has also suggested that a high TB harms anticancer immunity  in severIn our study, a high tumor SUVmax (>12) was not correlated with OS, either with PFS in ES-SCLC patients treated with CIT, or in patients treated with CT. These results are consistent with previous studies: a recent meta-analysis published in 2021 by Christensen , showed Moreover, we showed that liver metastasis was associated with a worse OS for ES-SCLC patients treated with CIT , but not for patients treated with CT. Patients with liver metastases often have more aggressive cancers, which also contribute to poorer responses to systemic therapies. Liver involvement is a well-known pejorative indicator that is associated with reduced responses and worse outcomes (PFS and OS) in patients treated with ICI . One reaA combination of multiple biomarkers could be a relevant way to optimize both prediction and prognostic accuracy ,25. We dMultiple previous studies had demonstrated that a high peripheral pro-inflammatory status, which can be measured using biomarkers such as LDH and dNLR, was associated with worse outcomes in patients with cancer ,27,28, aThe main limitations of our study are its retrospective nature and its relatively small sample size. We compared patients from a period before and after immunotherapy availability in this setting. Nevertheless, there was no major modification in the standard of care of ES-SCLC for the two cohorts. Furthermore, another strength was the use of predictiveness curves for testing the predictive capacity of candidate biomarkers that enables the most complete analysis. Indeed, predictiveness curves fit into an integrative approach which could give information about risk  but also classification performance (as well as ROC curves).A high TMTV on 18F-FDG PET/CT was correlated with a worse outcome for ES-SCLC patients undergoing first-line CIT. This finding suggests that TMTV on 18F-FDG PET/CT could be used as a prognostic biomarker, identifying patients who shall not benefit from ICIs. Moreover, the TMTV/LM signature could also help to stratify patients at baseline, and guide clinical decision-making for ES-SCLC patients before first-line therapy. Such findings could be taken into account in clinical trials, and they might need further validation through large, independent, and prospective cohorts.


COVID-19 is an ongoing, global pandemic caused by the novel, highly infectious SARS-CoV-2 virus. Efforts to mitigate the effects of SARS-CoV-2, such as mass vaccination and development of monoclonal therapeutics, require precise measurements of correlative, functional neutralizing antibodies that block virus infection. The development of rapid, safe, and easy-to-use neutralization assays is essential for faster diagnosis and treatment. Here, we developed a vesicular stomatitis virus (VSV)-based neutralization assay with two readout methods, imaging and flow cytometry, that were capable of quantifying varying degrees of neutralization in patient serum samples. We tested two different spike-pseudoviruses and conducted a time-course assay at multiple multiplicities of infection (MOIs) to optimize the assay workflow. The results of this assay correlate with the results of previously developed serology and surrogate neutralization assays. The two pseudovirus readout methods produced similar values of 50% neutralization titer values. Harvest-free in situ readouts for live-cell imaging and high-throughput analysis results for flow cytometry can provide unique capabilities for fast evaluation of neutralization, which is critical for the mitigation of future pandemics. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), emerged in December 2019 and resulted in a global pandemic [pandemic ,4. TMPRSpandemic . Broadlypandemic . To predSeveral methods to quantify nAb titers in patient serum have been established, including live virus, pseudovirus, and ELISA-based neutralization assays ,8. Use oTwo different pseudoviruses were tested, a lentivirus and a VSV-based pseudovirus with the original Wuhan-Hu-1-strain spike protein and GFP reporter. After 24 h at a multiplicity of infection (MOI) of 1.0, the VSV-based pseudovirus had the most GFP expression with an average of 80.9 ± 3.06% compared to the lentivirus at 26.6 ± 1.99% . Additiop = 0.067), and GFP expression at 16 h did not differ between the three MOIs tested (p = 0.299). However, the 16 h timepoint at an MOI of 0.5 was chosen for the neutralization assay because the difference between the timepoints and MOIs was insignificant. These assay parameters allowed for more manageable workflow timing and reduced the virus consumption. A small increase in dead cells was seen at the 24 h timepoint at an MOI of 2.0.To develop a rapid and easy-to-use neutralization assay, cells were infected with the pseudovirus to determine the optimal time and MOI for the assay. HEK293T-hACE-2-TMPRSS2-mCherry cells were infected with the VSV-spike pseudovirus at MOIs of 0.5, 1.0, or 2.0 for 2, 4, 8, 16, and 24 h . Maximump = 0.446), and a Spearman rank correlation test showed high correlation between the two assays.   B.s) showed significant correlation between the NT50 values of the pseudovirus assay and these assays  compared to the pseudovirus  and surrogate  assays  assays . Based op = 0.069) and the three MOIs at 16 h did not statistically differ (p = 0.299), the MOI of 0.5 and the 16 h timepoint were chosen.One goal for pandemic preparedness is to develop rapid, easy-to-use, high-throughput assays to help determine COVID-19 serological diagnosis and vaccination efficacy. Pseudovirus assays offer BSL-2 convenience and ease of use to laboratories measuring serology neutralization. When testing two pseudoviruses, we found that the VSV-based pseudovirus resulted in a higher percent GFP expression after 24 h than the lentivirus-based pseudovirus . After sWhen considering the pseudovirus assay readout methods, both live-cell imaging and flow cytometry readouts provided comparable measurements of 50% neutralization titer. Using a dual approach for determining the neutralization provided insight on optimizing samples for both flow cytometry and imaging. The mCherry reporter in the cell line was useful for the imaging readout by providing a fluorescence-based method to segment and normalize the total cells to the virus-infected cells that expressed GFP. Analysis of the total cells by imaging required the optimization of the mCherry segmentation parameters specific to this cell type, while the flow cytometry analysis based on scatter was more straightforward . The celp = 0.446), and the results were highly correlated, demonstrating potential to use these analysis methods interchangeably (s = 0.988). Additionally, these two orthogonal measurements of neutralization on the same pseudovirus-infected samples provided assurance that the measured percent infectivity and resulting pseudovirus neutralization titer was correct.Overall, the imaging method for this neutralization assay relied on a fluorescent reporter within the cell, a fibronectin coating, and care to ensure that no bubbles were introduced during the addition of the serum–virus mixture. The flow cytometry method required additional sample harvests and washes but had a straightforward gating strategy to quantify neutralization. The imaging method provided automated in situ cell measurements within the plate and automated image analysis, but the flow cytometry method was able to process more cells than were able to be imaged. Nine 10× magnification fields of view were used to image cells to avoid microplate well-edge effects. This area represented approximately 13% of the total well surface area and sampling of the cell population, whereas flow cytometry was able to analyze the entire cell population. Despite the smaller sampling by imaging, there were no significant differences between the NT50 values of the imaging and flow cytometry analyses (s = 0.7971 and rs = 0.8796 p < 0.0001). Higher anti-spike IgG titers correlated with higher NT50 values, indicating that the pseudovirus neutralization assay has potential to be benchmarked to a quantitative value (BAU/mL), enabling improved comparability of NT50 titers. Additionally, differences between the characteristics of these assays could result in low correlation for some of the samples. First, the pseudovirus assay was a cell-based assay, while the serology and surrogate assays were both bead-based assays. For the bead-based neutralization assay, the correlation showed that the NT50 values had a trend of being higher for the pseudovirus assay than the surrogate assay. One potential explanation for this was that the target antigen for the surrogate assay was the RBD protein compared to the pseudovirus containing a spike protein. It is likely that there are some neutralizing antibodies directed towards other regions of the spike not within the RBD domain [Serum samples processed via the pseudovirus neutralization assay were concurrently processed with the previously validated bead-based serology assay and bead-based surrogate neutralization assay . CompariIn summary, this work highlights a cell-based pseudovirus neutralization assay that is straightforward and easy to perform. The use of a pseudovirus offers a safer alternative to the live-virus neutralization assays, which require BSL-3 laboratories. The assay can be easily adapted using pseudoviruses of new SARS-CoV-2 variants and future pandemic viruses. The hands-off nature of the imaging readout and the high-throughput capability of the flow cytometry readout show fast and convenient ways to quantify neutralization, which is critical to future pandemic preparedness. The methods demonstrated here can serve an important role to quantify neutralizing antibody titers needed for the future development of antivirals and monoclonal therapeutics.HEK293T , HEK293T-hAce2 , and HEK293T-hAce2-TMPRSS2-mCherry  cells were grown in Eagle’s Minimum Essential Medium  + 10% heat-inactivated fetal bovine serum (HI-FBS) . All cells were grown with vendor-specified selection antibiotics in a T25 flask to 70–90% confluency after 2 to 3 days and were subsequently passaged. The cells were collected using Trypsin EDTA  and seeded at 1 million cells per flask. Cells were used between passages 3 and 20.2 for 24 h. HEK293T cells were used as a negative control. After 24 h, the average cell count for each well was determined after trypsinizing 10 wells on each plate using a Multisizer 3 Coulter Counter . To calculate a multiplicity of infection (MOI) of 1.0 for SARS-CoV2, SΔG-GFP Pseudotyped VSV  with a provided virus titer of 107 plaque-forming units per mL was used. For SARS-CoV-2 spike pseudotyped lentivirus with an eGFP reporter , a virus titer of 2 × 105 transducing units per mL was provided by the manufacturer and used to calculate the MOI. Both pseudoviruses had SARS-CoV-2 spike proteins from the Wuhan-Hu-1 strain. The MOI was calculated by multiplying the average cell count by the desired MOI and dividing by the virus titer provided by the manufacturer. The infection proceeded for 24 h before supernatant was collected, and attached cells were washed and trypsinized for 3–5 min. Trypsin was inactivated with media, and the cells and supernatant were combined and subsequently washed and run through the NxT Attune flow cytometer .To improve cell adhesion, 96-well tissue-culture-treated plates  were coated with 10 μg/mL of fibronectin  for 4–6 h before washing and seeding the cells. HEK293T-hAce2-TMPRRS2-mCherry cells were seeded at 7500 cells per well and allowed to adhere at 37 °C with a 5% volume fraction of CO2 for 24 h, and tested in the time-course study with three different MOIs of VSV-based pseudovirus: 0.5, 1.0, and 2.0. The MOI was calculated by trypsinizing the cells as described in HEK293T-hACE-2-TMPRSS2-mCherry cells were plated on a 96-well tissue-culture-treated plate at 7500 cells per well, placed in a 37 °C incubator with a 5% volume fraction of COSerum samples of convalescent, vaccinated, and pre-COVID patients were collected before 2021 and provided by the Frederick National Laboratory for Cancer Research of the National Cancer Institute, the Center for Disease Control, and Abbott Laboratories through respective material transfer agreements (MTAs). Seventy-seven serum samples were tested, including twenty-eight known negative samples . These s2 for 24 h. After 24 h of adhesion, an average cell count from 10 wells was used to calculate the MOI.As described in A 9-point curve to obtain 50% neutralization titer (NT50) was generated with an initial 5-fold dilution of serum in OPTI-MEM  + 2% HI-FBS, and then 8 subsequent 3-fold dilutions . VSV-basUsing the Cytation 5, brightfield, GFP, and mCherry fluorescence images were acquired using a 10x objective across nine fields of view per well. The nine fields of view were acquired in the center of the well to avoid well-edge effects. Imaging was performed every 75 min across all sample wells. Image processing and analysis were performed using a custom script implemented with MATLAB R2022a . GFP and mCherry images were segmented using the empirical gradient threshold method . The totg for 5 min and supernatants were removed. Cells were resuspended in PBS + 2% HI-FBS and put on a non-tissue-culture-treated 96-well plate for loading on the NxT Attune Autosampler. To set up fluorescence compensation, HEK293T-hAce2 cells were used as an unstained control. HEK293T-hAce2 cells that had been infected with VSV-ΔG spike pseudovirus for 24 h were used for GFP compensation, while HEK293T-hAce2-TMPRSS2-mCherry cells were used for mCherry compensation. Samples were analyzed using FlowJo v10.8.1  and gated for cells based on the side and forward scatter areas, single cells based on the forward scatter height and area, and GFP expression . For each wash, supernatants were collected. Wells were trypsinized for 3 to 5 min, the trypsin was inactivated with media, and each sample was diluted with PBS + 2% HI-FBS. Cells were spun at 200× pression . The GFPThe spike IgG serology assay and surrogate neutralization assay were performed as previously described . BrieflyFor the bead-based surrogate neutralization assay, patient serum samples were serially diluted and incubated with RBD (Wuhan-Hu-1 strain)-coated beads in a 96-well plate at 10,000 beads per well for 30 min, shaking at 750 rpm at RT in the dark. The plates were washed three times with PB-T after using a magnetic separator for 1 min. Biotinylated ACE-2 (0.625 µg/mL) was added to each well and incubated while shaking for 1 h. The beads were washed again and then incubated with PE-Streptavidin (0.6 µg/mL) for 30 min of shaking. The beads were washed and resuspended in PB-T and run on the Cytoflex S  flow cytometer. If neutralization occurred, lower serum dilutions resulted in lower fluorescence signals.All statistical analyses were conducted in GraphPad Prism 9 .For the time-course assay, a two-way analysis of variance (ANOVA) with multiple comparisons was used to determine if there was a statistically significant difference between the three MOIs tested across the different timepoints.GFP)Max − %GFP)/(%GFP)Max), where (%GFP)max is the control sample treated with pseudovirus without a serum sample. For the imaging analysis method, the reduction percentage was defined as Reduction% = 100 × ((Area%GFP)max − Area%GFP)/(Area%GFP)Max), where (Area%GFP)max is the control sample treated only with pseudovirus without a serum sample. The percent reduction was determined for each dilution. The NT50 was calculated using the variable slope model based on the Hill slope of the dilutions, and the NT50 was determined for both readout methods [For the pseudovirus neutralization assay, the percent GFP was used to calculate the percent reduction. For the flow cytometry analysis method, the reduction percentage was defined as Reduction% = 100 ×  curve was generated, including all samples for the three assays, to determine the sensitivity and specificity .


Aiming to promote the application of D-mannitol in the field of phase change thermal storage, obstacles, including low thermal storage efficiency and high supercooling, should be properly disposed of. The adoption of adaptable and low-cost supporting materials to make shape-stable phase change materials (ss-PCMs) affordable is a primary solution to solve the above shortcomings. In this study, high-performance ss-PCM for effective medium-temperature heat storage was prepared using expanded vermiculite as the support for D-mannitol preservation. Among the three candidates that treated the raw vermiculite by dilute acid, calcination, and microwave heating, the calcinated expanded vermiculite (CV) was characterized as the most suitable one. After impregnating D-mannitol into the CV carrier by vacuum, a melting enthalpy of 205.1 J/g and a crystallization enthalpy of 174.1 J/g were achieved by the as-received CV/D-mannitol ss-PCM. Additionally, the supercooling of the ss-PCM was reduced to 45.6 °C. The novel CV/D-mannitol ss-PCM also exhibited excellent reusability and stability. All the findings indicate that the abundant and inexpensive CV exhibited great potential as the supporting material for D-mannitol-based ss-PCMs, which allow effective waste heat recovery and temperature regulation. In facing the increasing unsustainable fossil fuel consumption and the large amounts of GHG emission, it is crucial to develop renewable energy systems for carbon peaking and carbon neutrality goals . HoweverD-mannitol is a renewable biochemical that possesses high heat storage density and stability, which can be potentially used as a PCM for medium-temperature energy storage ,6. Howev2/g, and the relative enthalpy efficiency of the as-formed ss-PCMs was 94.45%. In another work, Zhang et al. designed mica-stabilized polyethylene glycol (PEG) ss-PCMs [In the past decades, extensive research has been conducted for continuous exploration of the porous supporting materials candidates for the preservation of PCMs . Among t ss-PCMs . A maximVermiculite is a silicate mineral that consists of a layered structure . Due to In this study, different expanding strategies, including acid treatment, calcination, and microwave heating, were conducted to generate a series of expanded vermiculites with different structural properties. Then, the expanded vermiculites were used as the supporting materials for the preservation of D-mannitol and used as the ss-PCMs. The moderately swollen vermiculite carriers are expected to facilitate the process of applying D-mannitol for efficient heat storage in the medium-temperature domain. This study adds new possibilities for vermiculite to develop advanced materials for sustainable energy storage applications.2O2  was purchased from Tianjin Fuchen Chemical Reagent Co., Ltd., Tianjin, China.Vermiculite was purchased from Xinlong Vermiculite Co., Yuli, China. D-mannitol (98 wt%) and citric acid (90 wt%) were obtained from Macklin Co., Ltd., Shanghai, China. H2O2 solution (15% (v/v)). Next, 10.0 g of dried vermiculite was added to the mixture, and the slurry stood for 24 h at 30 °C. Then, after washing with deionized water until it reached a neutral pH (~7), the solid fraction was collected and dried, which was defended as the acid-modified vermiculite (AV). The calcinated expanded vermiculite (CV) was prepared by heating the raw material in a muffle furnace at 800 °C for 5 min. The microwave-expanded vermiculite (MV) was obtained by heating the vermiculite in a microwave at 800 W for 10 min.To prepare the acid-treated expanded vermiculite, the raw vermiculite was first sieved to obtain particles with an approximate diameter of 2 mm. Then, the sieved vermiculite was dried out at 60 °C. In parallel, 4.0 g of citric acid was dissolved into 50 mL of HThe ss-PCMs were prepared using a vacuum impregnation strategy . In a tyθ values ranging from 5° to 80° and a scan rate of 5°/min. The chemical functional diagram of the ss-PCMs and the compatibility of the carrier with D-mannitol were tested using a Fourier transform infrared spectroscopy  in the wave range of 4000–400 cm−1. Brunauer-Emmett-Teller  was used to characterize the specific surface area and pore size distribution of the expanded vermiculite. The absorption and release of heat and the phase transition temperatures were analyzed by a differential scanning calorimeter . The heating/cooling rate was 5 °C/min, and the testing range was from room temperature (~25 °C) to 180 °C, followed by hold at 180 °C for 3 min. The thermal stability of the specimens in an N2 atmosphere was tested by thermogravimetric analysis  with a scanning temperature range of 30–800 °C, and a ramping rate of 5 °C/min. The ss-PCM specimens were placed in an oven at 180 °C and observed for melting and leakage.Scanning electron microscopy  was used to characterize the morphology of the expanded vermiculites and the corresponding ss-PCM. The crystal structure of D-mannitol was investigated by X-ray diffraction  with scan 2Before being used as a supporting material for the D-mannitol based ss-PCM, the expanded vermiculites specimens that were treated by different methods were characterized. As shown in 2O and the peak at 1009 cm referred to the Si-O-Si and Si-O-Al functional bonds, which always appeared no matter the raw vermiculite treatment or not [The XRD patterns in t or not , suggest2/g and the largest average pore size of 27.5 nm  and the effectiveness of latent heat storage (E) are the two decisive thermal performance indicators for thermal storage by ss-PCMs [mH) and crystallization (ΔcH) can be determined by calculating the integral area of the peaks in DSC curves. Phase transition temperatures can be determined by examining the baseline and the tangent intersects of the curves. R and E can be calculated by the following equations.mHD-mannitol, and ΔcHD-mannitol, are the latent heat of D-mannitol in the melting and crystallization processes, respectively. ΔmHss-PCM, and ΔcHss-PCM, are the melting enthalpy and crystallization enthalpy of ss-PCMs, respectively.Thermal storage capacity  and ΔcH (170.5 J/g), which may likely be due to the large specific surface area and pore structures of CV  reflects its superior thermal storage capacity. Furthermore, CV/D-mannitol also exhibited the highest E value (74.9%) among the tested ss-PCMs with different expanded vermiculites.es of CV . The impT) can be calculated from the difference between the melting temperature (mT) and the temperature of crystallization (cT): ΔT = mT − cT. According to the results shown in R) and efficiency (E). Through analyzing the enthalpy of phase change and conducting a comprehensive comparison, CV is selected as the the optimal support material for impregnating D-mannitol among the candidates. The degree of supercooling (ΔThe thermal stability of CV/D-mannitol ss-PCM was further investigated. As shown in mH and ΔcH only decreased by 4.8% and 4.2%, respectively (In order to further analyze the long-term stability of the novel CV/D-mannitol ss-PCMs, we performed 100 cycles of heating and cooling progression. The XRD patterns and FTIR spectrum of the fresh and used ss-PCMs are shown in ectively . TherefomH of 195.9 J/g and ΔcH of 170.5 J/g, which also showed the capability to reduce the supercooling of D-mannitol to 45.6 °C, effectively alleviating the heat release delay phenomenon. Even after 100 cycles of heating/cooling cycles, the CV/D-mannitol can still maintain a high phase change enthalpy, showing good durability and suitability for long-term use. Therefore, CV/D-mannitol showed great promise in medium temperature heat storage.The expanded vermiculite obtained from raw vermiculite through dilute acid treatment, calcination, and microwave heating can be used as supporting materials for ss-PCMs. Among them, CV exhibits superior performances in impregnating D-mannitol compared to other materials. The CV/D-mannitol exhibited Δ